This invention relates to protection against and diagnosis of flaviviral infection. More specifically, the invention concerns recombinantly produced dimers of truncated flaviviral envelope protein secreted as mature proteins from eucaryotic cells and which induce high titer virus neutralizing antibodies believed to be important in protection against flaviviral infection and which are useful in diagnosis of infection by the virus.
The four serotypes of dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4) belong to the family Flaviviridae which also includes the Japanese encephalitis virus (JE), Tick-borne encephalitis virus (TBE), West Nile virus (WN), and the family prototype, Yellow fever virus (YF). Flaviviruses are small, enveloped viruses containing a single, positive-strand, genomic RNA. The envelope of flaviviruses is derived from the host cell membrane and is decorated with virally-encoded transmembrane proteins membrane (M) and envelope (E). While mature E protein and the precursor to M, prM, are glycosylated, the much smaller mature M protein is not. The E glycoprotein, which is the largest viral structural protein, contains functional domains responsible for cell surface attachment and intraendosomal fusion activities. It is also a major target of the host immune system, inducing virus neutralizing antibodies, protective immunity, as well as antibodies which inhibit hemagglutination.
Dengue viruses are transmitted to man by mosquitoes of the genus Aedes, primarily A. aegypti and A. albopictus. The viruses cause an illness manifested by high fever, headache, aching muscles and joints, and rash. Some cases, typically in children, result in a more severe forms of infection, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), marked by severe hemorrhage, vascular permeability, or both, leading to shock. Without diagnosis and prompt medical intervention, the sudden onset and rapid progression of DHF/DSS can be fatal.
Flaviviruses are the most significant group of arthropod-transmitted viruses in terms of global morbidity and mortality with an estimated one hundred million cases of dengue fever occurring annually (Halstead, 1988). With the global increase in population and urbanization especially throughout the tropics, and the lack of sustained mosquito control measures, the mosquito vectors of flavivirus have distributed throughout the tropics, subtropics, and some temperate areas, bringing the risk of flaviviral infection to over half the world""s population. Modern jet travel and human emigration have facilitated global distribution of dengue serotypes, such that now multiple serotypes of dengue are endemic in many regions. Accompanying this in the last 15 years has been an increase in the frequency of dengue epidemics and the incidence of DHF/DSS. For example, in Southeast Asia, DHF/DSS is a leading cause of hospitalization and death among children (Hayes and Gubler, 1992).
The flaviviral genome is a single strand, positive-sense RNA molecule, approximately 10,500 nucleotides in length containing short 5xe2x80x2 and 3xe2x80x2 untranslated regions, a single long open reading frame, a 5xe2x80x2 cap, and a nonpolyadenylated 3xe2x80x2 terminus. The complete nucleotide sequence of numerous flaviviral genomes, including all four DEN serotypes and YF virus have been reported (Fu et al., 1992; Deubel et al, 1986; Hahn et al, 1988; Osatomi et al., 1990; Zhao et al, 1986; Mackow et al., 1987; Rice et al., 1985). The ten gene products encoded by the single open reading frame are translated as a polyprotein organized in the order, capsid (C), premembrane/membrane (prM/M), envelope (E), nonstructural protein (NS) 1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5 (Chambers, et al. 1990). Processing of the encoded polyprotein is initiated cotranslationally, and full maturation requires both host and virally-encoded proteases. The sites of proteolytic cleavage in the YF virus have been determined by comparing the nucleotide sequence and the amino terminal sequences of the viral proteins. Subsequent to initial processing of the polyprotein, prM is converted to M during viral release (Wengler, G. et al., J Virol (1989) 63:2521-2526) and anchored C is processed during virus maturation (Nowak et al., Virology (1987) 156:127-137).
While all dengue viruses are antigenically related, antigenic distinctions exist which define the four dengue virus serotypes. Infection of an individual with one serotype does not apparently provide long-term immunity against the other serotypes. In fact, secondary infections with heterologous serotypes are becoming increasingly prevalent as multiple serotypes co-circulate in a geographic area. In general, primary infections elicit mostly IgM antibodies directed against type-specific determinants. On the other hand, secondary infection by a heterologous serotype is characterized by IgG antibodies that are flavivirus crossreactive. Dengue virus vaccine development is complicated by the observation that immunity acquired by infection with one serotype may in fact enhance pathogenicity by dengue virus of other types. Halstead (1982) demonstrated that anti-dengue antibodies can augment virus infectivity in vitro, and proposes that serotype crossreactive, non-neutralizing antibodies to E enhance infection in vivo, resulting in DHF/DSS (Halstead, 1981). This viewpoint is not however, universally accepted (Rosen, 1989). For example, Kurane et al (1991) proposed that dengue serotype-cross-reactive CD4+ CD8xe2x88x92 cytotoxic T cells (CTLs) specific for NS3 may contribute to the pathogenesis of DHF/DSS by producing IFN-xcex3 and by lysing dengue virus-infected monocytes. Recent evidence demonstrating that CTLs specific for E are not serotype-crossreactive may suggest that use of E subunit vaccines would not induce the potentially harmful cross-reactive CTL response (Livingston et al, 1994). Regardless of the mechanism for enhanced pathogenicity of a secondary, heterologous dengue viral infection, strategies employing a tetravalent vaccine should avoid such complications. Helpful reviews of the nature of the flaviviral diseases, the history of attempts to develop suitable vaccines, and structural features of flaviviruses in general as well as the molecular structural features of the envelope protein of flaviviruses are available (Halstead 1988; Brandt 1990; Chambers et al., 1990; Mandl et al., 1989; Henchal and Putnak, 1990; Putnak 1994; Rey et al., 1995).
Although many approaches to dengue virus vaccines have been pursued, there is no acceptable vaccine currently available. Until recently, the low titer of dengue virus grown in culture has made a killed vaccine impractical, and candidate live-attenuated dengue virus vaccine strains tested to date have proven unsatisfactory (see, e.g., Eckels et al, 1984; Bancroft et al, 1984; McKee et al, 1987), although live attenuated candidate vaccine strains continue to be developed and tested (Hoke et al, 1990; Bhamarapravati et al, 1987). The construction of several full-length infectious flavivirus clones (Rice et al., 1989; Lai et al., 1991; Sumiyoshi et al., 1992) has facilitated studies aimed at identifying the determinants of virulence in flaviviruses (Bray and Lai, 1991; Chen et al., 1995; Kawano et al., 1993). However, these studies are in preliminary stages and little information on virulence has been obtained. A similar approach to vaccine development in the poliovirus system, while extremely informative, has taken years.
In the absence of effective live attenuated or killed flavivirus vaccines, a significant effort has been invested in the development of recombinant, flaviviral subunit or viral-vectored vaccines. Many of the vaccine efforts which use a recombinant DNA approach have focused on the E glycoprotein. This glycoprotein is a logical choice for a subunit vaccine as it is exposed on the surface of the virus and is believed to be responsible for eliciting protective immunity as monoclonal antibodies directed against purified flaviviral E proteins are neutralizing in vitro and some have been shown to confer passive protection in vivo (Henchal et al., 1985; Heinz et al., 1983; Mathews et al., 1984; Hawkes et al., 1988; Kimuro-Kuroda and Yasui, 1988).
Although the primary amino acid sequence of flaviviral E glycoproteins are variable (45-80% identity), all have twelve conserved cysteine residues, forming six disulfide bridges, and nearly superimposable hydrophilicity profiles suggesting that they probably have similar secondary and tertiary structures. Recently, the structure of a soluble fragment of the tick-borne encephalitis (TBE) virus envelope glycoprotein was solved at 2 xc3x85 resolution (Rey et al., 1995). This analysis demonstrated that the envelope glycoprotein in its native form is a homodimer which presumably extends parallel to the virion surface. This dimer is formed by an anti-parallel association of the two envelope glycoproteins stabilized by polar interactions along the central region of the dimer, and by non-polar interactions at either end (FIG. 1). The dimer is slightly curved relative to the virion surface, perhaps conforming to the shape of the lipid envelope. The convex, external face contains the major immunogenic sites and the carbohydrate side chains. The carboxy terminus extends from the concave internal face down toward the membrane. Based upon sequence alignments and conservation of cysteine residues involved in disulfide bridges, the authors suggest that the TBE structure serves as a good model for all flavivirus envelopes. Therefore, recombinant soluble dengue E expressed as a dimer might induce a more potent antiviral response than monomeric E because it more closely resembles the natural envelope glycoprotein.
Recombinant flavivirus E glycoprotein has been expressed in several systems to date (See Putnak, 1994 for recent review). In general the systems have proven unsatisfactory for production of a cost-effective flavivirus vaccine due to limitations in antigen quality, quantity, or both. The following paragraphs highlight the major flavivirus vaccine efforts and summarize the results obtained to date.
Bioenvelope glycoproteins vary widely in primary, secondary, tertiary, and quaternary structure. Functional similarity does not necessarily imply structural similarity. To demonstrate the type of variation seen in viral envelope glycoproteins one need look no further than the structures of HIV envelope, Tick Borne Encephalitis (TBE) virus envelope (a flavivirus very similar to dengue), influenza virus hemagglutinin glycoprotein, and Semliki Forest Virus envelope (SFV; an alpha virus). In terms of primary structure, the envelope glycoproteins tend to be the most highly divergent of any viral gene and thus minimal sequence similarity exists even within groups of closely related viruses. As one looks at highly divergent viruses (e.g. HIV and TBE or dengue) the sequence similarity is almost non-existent. In addition, they vary significantly in terms of secondary, tertiary, and quaternary structure as well. As illustrated in Kwong, P. D. et al. Nature (1998) 393:648-659, the structure of the HIV gp120 envelope glycoprotein is quite globular in nature and in fact does not include a transmembrane domain. The membrane anchor function of the HIV envelpe glycoprotein is provided by another protein, gp41 which associates non-covalently as a heterodimer with the gp120 protein maintaining its association with the membrane. In contrast, the structure of the flavivirus TBE envelope glycoprotein (Rey, F. A. et al. Nature (1995) 375:291-298) demonstrated that it exhibits an elongated structure. However, in contrast to other viral envelope glycoproteins which also have an elongated structure (e.g. influenza virus hemagglutinin discussed below) the elongated structure lies parallel to the membrane in a rather flat presentation. In fact, the flavivirus envelope exists on the surface of the membrane as a homodimer with head to tail orientation of the two monomers and is anchored in the membrane by its own transmembrane domain. The structure of the envelope glycoproteins of influenza virus (hemagglutinin and neuraminidase), while also elongated in form, exist as spikes protruding from the membrane and include unique structural features such as a hinge region (Reviewed in Fields, B. N. and D. M. Knipe (eds.) Virology, 2nd ed., Raven Press, NY, 1990). The hemagglutinin spikes are formed by the association of three monomers in a triple-stranded coiled-coil structure markedly different from the head to tail dimer form of the TBE envelope. Finally, although the alphaviruses are relatively closely related to the flaviviruses, the structure of an alphavirus envelope glycoprotein also varies significantly from the structure described for flaviviruses (Helenius, A. Cell (1995) 81:651-653). The SFV envelope glycoproteins have been shown to form spikes which project 80 nm from the membrane surface and consist of three E1-E2 pairs. Thus, even for relatively closely related viruses, the envelope glycoproteins, while serving the same function, have markedly different structural properties.
These markedly different primary, secondary, tertiary, and quaternary structures affect heterologous expression characteristics. In fact, in contrast to HIV envelope glycoprotein which is expressed at reasonable efficiency in both the Chinese Hamster Ovary (CHO) cell expression system (Berman et al. J Virol (1989) 63:3489-98) and the Drosophila cell expressoin system (Culp et al.), the dengue virus envelope glycoprotein is not efficiently in CHO but is efficiently expressed in the Drosophila system. Expression levels of dengue envelope in CHO being less than 0.1 mg/L.
Recombinant flavivirus E glycoprotein has been expressed in several systems to date (See Putnak, 1994 for recent review). In general the systems have proven unsatisfactory for production of a cost-effective flavivirus vaccine due to limitations in antigen quality, quantity, or both. The following paragraphs highlight the major flavivirus vaccine efforts and summarize the results obtained to date.
Most efforts using Escherichia coli have yielded poor immunogen incapable of eliciting neutralizing antibodies in mice. This may reflect non-native conformation of flavivirus proteins expressed by bacteria and the necessity to process the viral proteins through the secretion pathway in order to achieve proper do sulfide bond formation and glycosylation. Expression of dengue proteins using the eucaryotic yeasts Saccharomyces cerevisiae and Pichia pastoris results in less than desirable quantities of immunogenic recombinant product obtained. The expression levels of dengue E achieved in these systems are well below that which would be required to produce a cost-effective flavivirus vaccine. (John Ivy et al., unpublished data. Expression of 80% E in the above-mentioned yeast systems and fungal systems (Neurospora crassa) gave products that were highly glycosylated (contain extensive high mannose chains) which interferes with immunogenicity. Also, the yields were quite low (ranging from about 10-100 ng/ml (despite the ability of these systems to produce high yields generally).)
Attempts to express 80% E in the Chinese Hamster Ovary (CHO) cells expression system were particularly disappointing. Predictions that this mammalian expression normally infects mammals and the system supports all the necessary post-translational modifications required to get native confirmation, were wrong. In fact the yields were poorest of any system (less than 0.1 xcexcg/ml) and the Dengue envelope gene was completely unstable in this expression system.
Use of the baculovirus expression system for flavivirus subunit vaccine production has met with limited success (Reviewed in Putnak, Modern Vaccinology, 1994). In contrast to the high expression levels reported for various heterologous proteins in the baculovirus system, the levels of expression of flavivirus structural proteins were quite low (e.g. 5-10 xcexcg DEN-2 E/106 cells; Deubel et al., 1991), and reactivity against a panel of anti-flaviviral monoclonal antibodies (MAbs) indicated that many conformationally sensitive epitopes were not present (Deubel et al., 1991). This suggests that folding of recombinant E produced in the baculovirus system may differ from the natural viral E protein. Furthermore, immunization with baculovirus-expressed recombinant envelope protein from DEN-1 (Putnak et al., 1991), Japanese Encephalitis virus (McCown et al., 1990), or Yellow Fever virus (Despres et al., 1991) failed to elicit substantial titers of virus neutralizing antibodies or protection against viral challenge in mice.
Several reports have described vaccinia-flavivirus recombinants expressing envelope proteins as part of a polyprotein. The most consistently successful results in vaccinia expression of flaviviral proteins have been obtained co-expressing prM and E. Mice immunized with recombinant vaccinia expressing Japanese Encephalitis (JE) virus prM and E developed higher neutralizing antibody titers and survived higher challenge doses of virus ( less than 10,000 LD50; Konishi et al, 1992) than mice immunized with recombinant vaccinia virus expressing E alone ( less than 10 LD50; Mason et al, 1991). Similarly, mice immunized with a vaccinia-Yellow Fever (YF) virus recombinant expressing prM-E were protected from virus challenge at levels equivalent to that of the attenuated YFV-17D vaccine, while vaccinia-YF virus recombinants expressing E-NS1, C-prM-E-NS1, or NS1 failed to protect mice (Pincus et al, 1992). Vaccinia DEN-1 recombinants expressing prM-E elicited neutralizing and hemagglutination inhibiting antibodies in mice, while recombinants expressing DEN-1 C-prM-E-NS1-NS2a-NS2b elicited no E-specific immune response (Fonseca et al, 1994).
Coordinate synthesis of prM and E appears to be important to obtain the native conformation of E. Expression of E in the absence of prM may result in a recombinant product that presents a different set of epitopes than those of the native virion (Konishi and Mason 1993; Heinz et al, 1994; Matsuura et al, 1989). Epitope mapping of the E expressed with prM suggests that the co-expressed protein more closely resembles the native virus. As prM and E appear to form heterodimers during viral maturation and E undergoes an acid pH-induced conformational change, Heinz et al (1994) has suggested the association of prM and E is required to prevent irreversible pH-induced conformational changes during transit through the secretory pathway. However, it has been shown that carboxy-truncated forms of flavivirus E expressed in the absence of prM elicit protection from challenge (Men et al, 1991; Jan et al, 1993; Coller et al., in preparation), suggesting expression of E in the absence of prM can result in the display of protective epitopes.
Within the last ten years an alternative eucaryotic expression system which uses the Drosophila melanogaster Schneider 2 (S2) cell line has been developed and used to efficiently express the envelope glycoprotein of Human Immunodeficiency Virus (Ivey-Hoyle et al., 1991; Culp et al, 1991; van der Straten et al, 1989). We have applied this system to production of recombinant flavivirus subunit polypeptides and have found the system can easily produce 20-30 mg of recombinant protein per liter of medium (unpublished). The recombinant product we have focused most of our efforts on is a soluble form of flaviviral E, which is truncated at the carboxy-terminal end resulting in a polypeptide which represents approximately 80% of the full-length E molecule (amino acids 1-395; 80% E). We have expressed 80% E as a single open-reading frame with prM to enhance proper folding and secretion as described above. The expression levels achieved using this combination of expression system and recombinant DNA construct far exceed those achieved in other systems and does provide a cost-effective source of flaviviral antigen for vaccine production. In addition, we have demonstrated that the recombinant 80% E product secreted by these cells is capable of inducing neutralizing antibodies and protection in mice (Coller et al., in preparation.)
In two instances, however, applicants failed in their attempt to produce envelope glycoproteins in the Drosophila expression system. First, a 100-amino acid polypeptide which is a unique domain (Domain B; amino acids 296-395 of DEN-2E) within the 80% E molecule was expressed poorly in the Drosophila expression system. The expression levels for Domain B were significantly lower (less than 1 mg/l) than those achieved with 80% E (approximately 15 mg/l). Domain B was the most likely expressed polypeptide in S. cereviseae and P. pastoris which we evaluated with expression levels up to 575 mg/l for Domain B expressed in P. pastoris (compared to expression levels of approximately 1 mg/l for 80% E). Second, a truncated version of the measles hemagglutinin protein (90% HA) was expressed and secreted at very low levels in the Drosophila expression system (about 0.5 mg/l). Like dengue, measles has been refractory to stable expression in many systems (Hirano, A. et al. xe2x80x9cGeneration of mammalian cells expressing stably measles virus protein via bicistronic RNA,xe2x80x9d Journal of Virological Methods (1991) 33:135-147).
The two examples above show that protein expression is highly unpredictable (Goeddel, D. V. xe2x80x9cSystems for Heterologous Gene Expression,xe2x80x9d in Methods in Enzymology, Vol. 185, pp. 3- Academic Press, Inc., 1990). In this case, protein expression is further complicated by the complexity of expressing bioenvelope glycoproteins (Mustilli, A. C. et al. xe2x80x9cComparison of secretion of a hepatitis C virus glycoprotein in Saccharomyces cerevisiae and Kluyveromyces lactis,xe2x80x9d Res Microbiol (1999) 150:179-187).
Over the past approximately eight years of research relating to dengue 80% E, the assignee of the present application has spent over $6.5 million to arrive at the invention.
While the use of this combination of Drosophila S2 cells and prM80% E has allowed significant progress towards the production of an effective flavivirus vaccine, the ability of a small polypeptide, with limited antigenic complexity, to induce long term, protective immunity in a large, outbred population may be limited. Numerous studies have demonstrated that immunogenicity is directly related both to the size of the immunogen and to the antigenic complexity of the immunogen. Thus, in general, larger antigens make better immunogens. In addition, the structure of TBE envelope protein was recently solved (Rey et al., 1995) and this analysis revealed that the native form of E protein found on the surface of the virion is a homodimer (FIG. 1). Our recombinant flaviviral E protein discussed above is monomeric and therefore is not identical to the natural viral E protein. Thus, in an attempt to produce a recombinant flavivirus vaccine with enhanced immunogenicity we engineered several constructs designed to promote dimerization of the soluble 80% E which is so efficiently produced in the Drosophila cells. By enhancing dimerization we increase the potency of the vaccine by increasing the structural similarity to native, virally expressed E, as well as by increasing the size and antigenic complexity of the immunogen.
Several of the approaches we have adopted to enhance dimerization of soluble 80% E were originally developed for antibody engineering. Flexible peptide linkers have been used to link the variable heavy and variable light chain polypeptides in the engineering of single chain Fv""s (scFv; Huston et al., 1988; Bird et al., 1988). These linkers, which are often repeated GlyGlyGlyGlySer (Gly4Ser) unit (SEQ ID NO:1), exhibit limited torsional constraints on the linked polypeptides, and therefore offer a reasonable option for covalently connecting the carboxy end of one 80% E moiety to the amino terminus of the second 80% E moiety. Based on the distance from the carboxy terminus of one subunit and the amino terminus of the other in the crystal structure of TBE 80% E dimers (F. Heinz, personal communication), we designed a peptide linker, made up predominantly of Gly4Ser repeats, to link the two 80% E molecules. The linker was designed to be slightly longer than the distance in the native molecule, in order to avoid torsional constraint on the association of the two 80% E moieties.
The second and third approaches to engineer 80% E dimers used strategies developed to engineer self-associating scFv miniantibodies. For homodimer miniantibody expression, Pack et al. (1992; 1993) expressed the scFv as a fusion with a flexible linker hinge and one of two dimerization domains (FIG. 2). One dimerization domain was a parallel coiled-coil helix of a leucine zipper from the yeast GCN4 gene product (Landschulz et al., 1988; O""Shea et al., 1989). The other domain was two alpha helices spaced by a sharp turn that associate to form a homodimeric four-helix bundle (Ho and DeGrado, 1987). The hinge region used to link the dimerization domains to the scFv was taken from an antibody hinge region to achieve maximum rotational flexibility. When these antibody-hinge-helix constructs were expressed in E. coli, homodimer miniantibodies spontaneously formed and could be extracted from the soluble protein fraction of cell lysates. These antibodies were indistinguishable from whole antibodies in functional affinity. To express secreted 80% E that can spontaneously dimerize, we have used these dimerization domains connected to the 80% E domains by a flexible Gly4Ser tether.
The present invention discloses and claims vaccines containing, as an active ingredient, a secreted recombinantly produced dimeric form of truncated flaviviral envelope protein. The vaccines are capable of eliciting the production of neutralizing antibodies against flavivirus. In the illustrations below, the dimeric forms of truncated flaviviral envelope protein are formed 1) by directly linking two tandem copies of 80% E in a head to tail fashion via a flexible tether; 2) via the formation of a leucine zipper domain through the homodimeric association of two leucine zipper helices each fused to the carboxy terminus of an 80% E molecule; or 3) via the formation of a non-covalently associated four-helix bundle domain formed upon association of two helix-turn-helix moieties each attached to the carboxy terminus of an 80% E molecule. All products are expressed as a polyprotein including prM and the modified 80% E products are secreted from Drosophila melanogaster Schneider 2 cells using the human tissue plasminogen activator secretion signal sequence (tPAL). Secreted products are generally more easily purified than those expressed intracellularly, facilitating vaccine production.
One embodiment of the present invention is directed to a vaccine for protection of a subject against infection by a Flavivirus. The vaccine contains, as active ingredient, the dimeric form of truncated envelope (E) protein of a flaviviral serotype, for example a dengue virus serotype. The dimeric truncated E is secreted as a recombinantly produced protein from eucaryotic cells. The vaccine may further contain portions of additional flaviviral serotype dimeric E proteins similarly produced. A preferred embodiment of the present invention relates to a vaccine for the protection of a subject against infection by a dengue virus. The vaccine contains a therapeutically effective amount of a dimeric 80% E, where, the 80% E has been secreted as a recombinantly produced protein from eucaryotic cells, such as Drosophila cells. Further, the xe2x80x9c80% Exe2x80x9d refers in one instance to a polypeptide which spans from Met 1 to Gly 395 of the DEN-2 envelope protein. The sequences described in the present application represent the envelope protein from dengue type 2 virus; three additional distinct dengue serotypes have been recognized. Therefore, xe2x80x9c80% Exe2x80x9d also refers to the corresponding peptide region of the envelope protein of these serotypes, and to any naturally occurring variants, as well as corresponding peptide regions of the envelope (E) protein of other flaviviruses. For example, serotypes of dengue virus such as: DEN-1; DEN-2; DEN-3; and DEN-4, as well as serotypes of: Japanese encephalitis virus (JE), Tick-borne encephalitis virus (TBE), West Nile virus (WN), and the family prototype, Yellow fever virus (YF).
Other embodiments of the present invention are directed to three basic approaches for the construction of dimeric 80% E molecules. (See infra.) These include: linked 80% E dimer; 80% E ZipperI; 80% E ZipperII; and 80% E Bundle.
Still other embodiments of the present invention are directed to vaccines containing truncated envelope protein of dimeric 80% E of more than one serotype to form multivalent vaccines, (i.e., divalent, trivalent, tetravalent, etc.). For example, such embodiments of the present invention include: a vaccine containing a first dimeric 80% E product of one flaviviral serotype and a second dimeric 80% E product of a second flaviviral serotype, and a third dimeric 80% E product of a third flaviviral serotype and a fourth dimeric 80% E product of a fourth flaviviral serotype, as well as in combination with other dimeric 80% E, each of a separate serotype one from another, where all dimeric 80% Es have been secreted as recombinantly produced protein from eucaryotic cells, such as Drosophila cells. It is considered that the present invention clearly includes vaccines that are comprised of multivalent truncated envelope protein of dimeric 80% E, which embrace two, three, four or more serotypes. For example, these serotypes may include the following dengue virus serotypes: DEN-1; DEN-2; DEN-3; and DEN-4, as well as other flavivirus serotypes of: Japanese encephalitis virus (JE), Tick-borne encephalitis virus (TBE), West Nile virus (WN), and the family prototype, Yellow fever virus (YF).
Additional embodiments of the present invention contemplate compositions of antibodies consisting essentially of antibodies generated in a mammalian subject administered an immunogenic amount of a vaccine containing dimeric 80% E as well as containing a first dimeric 80% E and a second dimeric 80% E, where both first and second dimeric 80% E have been secreted as recombinantly produced protein from eucaryotic cells, such as Drosophila cells. These vaccines could include multivalent truncated envelope protein of dimeric 80% E, which embrace two, three, four or more serotypes. These serotypes may include dengue virus serotypes: DEN-1; DEN-2; DEN-3; and DEN-4, as well as serotypes of: Japanese encephalitis virus (JE), Tickborne encephalitis virus (TBE), West Nile virus (WN), and the family prototype, Yellow fever virus (YF).
Still other embodiments of the present invention are drawn to immortalized B cell lines, where the B cells have been generated in response to the administration to a mammalian subject of an immunogenic amount of a vaccine containing truncated envelope protein of dimeric 80% E of more than one serotype to form multivalent vaccines, (i.e., divalent, trivalent, tetravalent, etc.). For example, such embodiments of the present invention include: a vaccine containing a first dimeric 80% E product of one flaviviral serotype and a second dimeric 80% E product of a second flaviviral serotype, and a third dimeric 80% E product of a third flaviviral serotype and a fourth dimeric 80% E product of a fourth flaviviral serotype, as well as in combination with other dimeric 80% E, each of a separate serotype one from another, where all dimeric 80% Es have been secreted as recombinantly produced protein from eucaryotic cells, such as Drosophila cells. These vaccines could include multivalent truncated envelope protein of dimeric 80% E, which embrace two, three, four or more serotypes. These serotypes may include dengue virus serotypes: DEN-1; DEN-2; DEN-3; and DEN-4, as well as serotypes of: Japanese encephalitis virus (JE), Tick-borne encephalitis virus (TBE), West Nile virus (WN), and the family prototype, Yellow fever virus (YF).
Further embodiments of the present invention are drawn to monoclonal antibodies secreted by these immortalized B cell lines.
Still further embodiments of the present invention are drawn to methods to protect a subject against a Flavivirus. These methods include the step of administering in a suitable manner to a subject in need of such protection an effective amount of a vaccine containing dimeric 80% E on a schedule optimum for eliciting such a protective immunoreactive response.
Another embodiment of the present invention is directed to methods to utilize the dimeric form of truncated flavivirus envelope protein for diagnosis of infection in individuals at risk for the disease. The diagnostic contains, as active ingredient, the dimeric form of truncated envelope protein of a flavivirus serotype. The dimeric truncated E is secreted as a recombinantly produced protein from eucaryotic cells. The diagnostic may further contain portions of additional flavivirus serotype dimeric E proteins similarly produced.
A preferred embodiment of the present invention relates to an immunodiagnostic for the detection of a Flavivirus, where the immunodiagnostic contains, a dimeric 80% E that has been secreted as a recombinantly produced protein from eucaryotic cells, such as Drosophila cells. Specifically, a preferred embodiment of the present invention relates to an immunodiagnostic for the detection of a flavivirus. Embodiments of the present invention include immunodiagnostics for the detection of a Flavivirus, where the immunodiagnostic contains, dimeric 80% E of more than one serotype to form multivalent immunodiagnostics, (i.e., divalent, trivalent, tetravalent, etc.). For example, such embodiments of the present invention include: an immunodiagnostics containing a first dimeric 80% E product of one flaviviral serotype and a second dimeric 80% E product of a second flaviviral serotype, and a third dimeric 80% E product of a third flaviviral serotype and a fourth dimeric 80% E product of a fourth flaviviral serotype, as well as in combination with other dimeric 80% E, each of a separate serotype one from another, where all of the dimeric 80% Es have been secreted as recombinantly produced protein from eucaryotic cells, such as Drosophila cells.
The present invention includes the embodiments of immunodiagnostic kits for the detection of a Flavivirus, in a test subject. These immmunodiagnostic kits contain (a) dimeric 80% E, where the dimeric 80% E has been secreted as recombinantly produced protein from eucaryotic cells, such as Drosophila cells; (b) a suitable solid support phase coated with dimeric 80% E; and (c) labeled antibodies immunoreactive to antibodies from the test subject.
Other embodiments of the immunodiagnostic kits of the present invention include multivalent dimeric 80% E of more than one serotype to form multivalent immunodiagnostics, (i.e., divalent, trivalent, tetravalent, etc.). For example, such embodiments of the present invention include: an immunodiagnostics containing a first dimeric 80% E product of one flaviviral serotype and a second dimeric 80% E product of a second flaviviral serotype, and a third dimeric 80% E product of a third flaviviral serotype and a fourth dimeric 80% E product of a fourth flaviviral serotype, as well as in combination with other dimeric 80% E products, each of a separate serotype one from another, where all of the dimeric 80% E products have been secreted as recombinantly produced protein from eucaryotic cells, such as Drosophila cells.
Further embodiments of the present invention relate to compositions of matter, that include a vector host recombinant DNA expression system, containing: (a) a suitable eucaryotic host cell; (b) a suitable recombinant DNA expression vector; (c) DNA encoding dimeric 80% E, operably linked and under the control of a suitable promoter; and (d) where the DNA encoding dimeric 80% E is also operably linked to a secretory signal leader sequence. The present invention further includes embodiments of a vector host recombinant DNA system where the dimeric 80% E is selected from the group consisting of: linked 80% E dimer; 80% E ZipperI; 80% E ZipperII; and 80% E Bundle. A preferred embodiment of the present invention relates to a vector host recombinant DNA system where the eucaryotic host cell is a Drosophila cell.
Other compositions of matter embodied in the present invention include DNA sequences encoding dimeric 80% E, specifically including DNA sequences encoding: linked 80% E dimer; 80% E ZipperI; 80% E ZipperII; and 80% E Bundle.